Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein.

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Identification of clinically approved small molecules that inhibit growth and affect transcript levels of developmentally regulated genes in the African trypanosome.

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. We screened a library of 1,585 U.S. or foreign-approved drugs and identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wished to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites lack the immune evasion mechanisms prevalent in bloodstream forms, making them vulnerable to the host immune system. To identify drugs that increase transcript levels of an invariant, insect-stage specific surface protein called procyclin, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction or stabilization of the procyclin transcript. Using these bloodstream reporter strains in combination with automated flow cytometry, we identified eflornithine, spironolactone, and phenothiazine as small molecules that increase abundance of procyclin transcript. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. While we failed to identify compounds that increase levels of procyclin protein on the cell surface, this study is proof of principle that these fluorescent reporter parasites represent a useful tool for future small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions.

Curcumin mediates polyamine metabolism and sensitizes gastrointestinal cancer cells to antitumor polyamine-targeted therapies.

Curcumin, a natural polyphenol that contributes to the flavor and yellow pigment of the spice turmeric, is known for its antioxidant, anti-inflammatory, and anticarcinogenic properties. Capable of affecting the initiation, promotion, and progression of carcinogenesis through multiple mechanisms, curcumin has potential utility for both chemoprevention and chemotherapy. Previous studies demonstrated that curcumin can inhibit ornithine decarboxylase (ODC) activity in human leukemia and breast cancer cells, and pretreatment with dietary curcumin blocks carcinogen-induced ODC activity in rodent models of skin, colon, and renal cancer. The current study investigated the regulation of polyamine metabolism in human gastric and colon carcinoma cell lines in response to curcumin. Curcumin treatment significantly induced spermine oxidase (SMOX) mRNA and activity, which results in the generation of hydrogen peroxide, a source of ROS. Simultaneously, curcumin down regulated spermidine/spermine N1-acetyltransferase (SSAT) activity and the biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase (SAMDC), thereby diminishing intracellular polyamine pools. Combination treatments using curcumin with the ODC inhibitor 2-difluoromethylornithine (DFMO), an agent currently in clinical chemoprevention trials, significantly enhanced inhibition of ODC activity and decreased growth of GI cancer cell lines beyond that observed with either agent alone. Similarly, combining curcumin with the polyamine analogue bis(ethyl)norspermine enhanced growth inhibition that was accompanied by enhanced accumulation of the analogue and decreased intracellular polyamine levels beyond those observed with either agent alone. Importantly, cotreatment with curcumin permitted the lowering of the effective dose of ODC inhibitor or polyamine analogue. These studies provide insight into the polyamine-related mechanisms involved in the cancer cell response to curcumin and its potential as a chemopreventive or chemotherapeutic agent in the GI tract.

Atractylenolide I stimulates intestinal epithelial repair through polyamine-mediated Ca2+ signaling pathway.

BACKGROUND: An impairment of the integrity of the mucosal epithelial barrier can be observed in the course of various gastrointestinal diseases. The migration and proliferation of the intestinal epithelial (IEC-6) cells are essential repair modalities to the healing of mucosal ulcers and wounds. Atractylenolide I (AT-I), one of the major bioactive components in the rhizome of Atractylodes macrocephala Koidz. (AMR), possesses multiple pharmacological activities. This study was designed to investigate the therapeutic effects and the underlying molecular mechanisms of AT-I on gastrointestinal mucosal injury. METHODS: Scratch method with a gel-loading microtip was used to detect IEC-6 cell migration. The real-time cell analyzer (RTCA) system was adopted to evaluate IEC-6 cell proliferation. Intracellular polyamines content was determined using high performance liquid chromatography (HPLC). Flow cytometry was used to measure cytosolic free Ca2+ concentration ([Ca2+]c). mRNA and protein expression of TRPC1 and PLC-γ1 were determined by real-time PCR and Western blotting assay respectively. RESULTS: Treatment of IEC-6 cells with AT-I promoted cell migration and proliferation, increased polyamines content, raised cytosolic free Ca2+ concentration ([Ca2+]c), and enhanced TRPC1 and PLC-γ1 mRNA and protein expression. Depletion of cellular polyamines by DL-a-difluoromethylornithine (DFMO, an inhibitor of polyamine synthesis) suppressed cell migration and proliferation, decreased polyamines content, and reduced [Ca2+]c, which was paralleled by a decrease in TRPC1 and PLC-γ1 mRNA and protein expression in IEC-6 cells. AT-I reversed the effects of DFMO on polyamines content, [Ca2+]c, TRPC1 and PLC-γ1 mRNA and protein expression, and restored IEC-6 cell migration and proliferation to near normal levels. CONCLUSION: Our data demonstrate that AT-I stimulates intestinal epithelial cell migration and proliferation via the polyamine-mediated Ca2+ signaling pathway. Therefore, AT-I may have the potential to be further developed as a promising therapeutic agent to treat diseases associated with gastrointestinal mucosal injury, such as inflammatory bowel disease and peptic ulcer.

Difluoromethylornithine in cancer: new advances.

Difluoromethylornithine (DFMO; eflornithine) is an irreversible suicide inhibitor of the enzyme ornithine decarboxylase which is involved in polyamine synthesis. Polyamines are important for cell survival, thus DFMO was studied as an anticancer agent and as a chemoprevention agent. DFMO exhibited mainly cytostatic activity and had single agent efficacy as well as activity in combination with other chemotherapeutic drugs for some cancers and leukemias. Herewith, we summarize the current knowledge of the anticancer and chemopreventive properties of DFMO and assess the status of clinical trials.

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression.

PURPOSE: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. EXPERIMENTAL DESIGN: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. RESULTS: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. CONCLUSIONS: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391-404. ©2016 AACR.

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

Catalase therapy corrects oxidative stress-induced pathophysiology in incipient diabetic retinopathy.

PURPOSE: Preclinical studies have highlighted retinal oxidative stress in the pathogenesis of diabetic retinopathy. We evaluated whether a treatment designed to enhance cellular catalase reduces oxidative stress in retinal cells cultured in high glucose and in diabetic mice corrects an imaging biomarker responsive to antioxidant therapy (manganese-enhanced magnetic resonance imaging [MEMRI]). METHODS: Human retinal Müller and pigment epithelial cells were chronically exposed to normal or high glucose levels and treated with a cell-penetrating derivative of the peroxisomal enzyme catalase (called CAT-SKL). Hydrogen peroxide (H2O2) levels were measured using a quantitative fluorescence-based assay. For in vivo studies, streptozotocin (STZ)-induced diabetic C57Bl/6 mice were treated subcutaneously once a week for 3 to 4 months with CAT-SKL; untreated age-matched nondiabetic controls and untreated diabetic mice also were studied. MEMRI was used to analytically assess the efficacy of CAT-SKL treatment on diabetes-evoked oxidative stress-related pathophysiology in vivo. Similar analyses were performed with difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. RESULTS: After catalase transduction, high glucose-induced peroxide production was significantly lowered in both human retinal cell lines. In diabetic mice in vivo, subnormal intraretinal uptake of manganese was significantly improved by catalase supplementation. In addition, in the peroxisome-rich liver of treated mice catalase enzyme activity increased and oxidative damage (as measured by lipid peroxidation) declined. On the other hand, DFMO was largely without effect in these in vitro or in vivo assays. CONCLUSIONS: This proof-of-concept study raises the possibility that augmentation of catalase is a therapy for treating the retinal oxidative stress associated with diabetic retinopathy.

Anticarcinogenic activity of alpha-difluoromethylornithine, ginseng, eleutherococcus, and leuzea on radiation-induced carcinogenesis in female rats.

PURPOSE: To carry out a comparative study of inhibition of radiation carcinogenesis using alpha-difluoromethylornithine (DFMO), tinctures of ginseng, eleutherococcus and leuzea in female rats. MATERIALS AND METHODS: Locally bred female LIO-strain rats were subjected to a single whole body γ-irradiation dose of 4 Gy at 12 weeks of age. Modifying drugs were given with drinking water from the 10th day after irradiation until the end of the experiment (for 16 months). RESULTS: Irradiated rats developed tumors 70.0-79.6% (malignant tumors: 43.7-45.0%) with a multiplicity of 1.48-1.75 (malignant: 0.5-0.58), while in unirradiated animals the incidence of all/malignant tumors was 21.9%/7.7% with multiplicity of 0.22/0.08. In exposed rats tumors most often developed in the mammary gland - 57.3%, reproductive and endocrine organs - 27.2%, and other localizations - 29.1%. All drugs, except leuzea, significantly reduced incidence and multiplicity of tumors, overall or at some localizations in irradiated rats. Highest, and practically equal inhibition, was shown by ginseng and DFMO, while eleutherococcus was clearly inferior. Ginseng reduced overall tumor incidence and multiplicity by 1.5 and 2.4 times, malignant tumor incidence and multiplicity - by 2.5 and 2.9 times, respectively. CONCLUSIONS: The ginseng extract is the most promising radiation carcinogenesis inhibitor tested.

Unbiased metabolite profiling indicates that a diminished thymidine pool is the underlying mechanism of colon cancer chemoprevention by alpha-difluoromethylornithine.

UNLABELLED: The ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO) is a highly effective chemopreventive agent for colorectal cancer thought to act via polyamine depletion. However, in DFMO-treated patients, mucosal polyamine levels do not directly correlate with colorectal cancer risk. Untargeted metabolite profiling was used to broadly survey DFMO actions on colon cancer cell metabolism. We found that DFMO treatment of Apc(Min) intestinal tumors and human colorectal cancer cells is associated with reduced levels of folate-dependent metabolites, including S-adenosylmethionine (SAM), thymidine pools, and related pathway intermediates. We hypothesized that unrestrained SAM consumption/regeneration constitutes a futile DFMO-triggered cascade that can steal tetrahydrofolate from thymidylate synthase and thereby diminish thymidine pools. In accord with this hypothesis, DFMO treatment altered the folate cofactor balance and thymidine supplementation prevented DFMO-elicited cytostasis without restoring polyamine levels. These findings suggest that thymidine metabolite pool insufficiency is a fundamental mechanism of DFMO cytostatic activity. SIGNIFICANCE: A previously unappreciated metabolic linkage between polyamine and thymidine biosynthesis is revealed, based on the competing requirement of these pathways for a limited pool of tetrahydrofolate cofactor. This study identifies the fi rst shared mechanism for colorectal cancer chemoprevention and chemotherapy, suggesting a common metabolic target for both premalignant and malignant colon cells.

Arginine enhances embryo implantation in rats through PI3K/PKB/mTOR/NO signaling pathway during early pregnancy.

Our previous study has demonstrated that dietary arginine supplementation during early pregnancy enhanced embryo implantation in rats. However, the mechanism was not clear. The objective of this study was to determine the mechanism that arginine enhanced embryo implantation during early pregnancy. Rats were fed the basal diets supplemented with 1.3% (wt:wt) L-arginine-HCl or 2.2% (wt:wt) L-alanine (isonitrogenous control) once pregnancy. On d4 of pregnancy, rats were given intrauterine injection of L-NG-nitro arginine methyl ester (L-NAME, nitric oxide synthase inhibitor), α-difluoromethylornithine (DFMO, polyamine synthesis inhibitor), wortmannin (PI3K inhibitor), or rapamycin (mTOR inhibitor). On d7 of pregnancy, rats were killed. Intrauterine injection of L-NAME decreased the implantation sites, while dietary arginine supplementation increased the implantation sites. Intrauterine injection of DFMO decreased the pregnancy rate, which was reversed by dietary arginine supplementation. Intrauterine injection of rapamycin or wortmannin inhibited embryo implantation. However, dietary arginine supplementation did not reverse this inhibition. Western blot analysis revealed that the expression of uterine p-PKB and p-S6K1 was greater in rats fed the arginine-supplemented diet in the presence of L-NAME treatment compared with rats fed the control diet. In the presence of DFMO treatment, the expression of uterine iNOS and eNOS was significantly enhanced in the arginine group compared with the control group. Similarly, intrauterine injection of wortmannin or rapamycin decreased the expression of uterine iNOS and eNOS, which was enhanced by dietary arginine supplementation. These data indicated that dietary arginine supplementation during early pregnancy could enhance embryo implantation through stimulation of PI3K/PKB/mTOR/NO signaling pathway.

HuR-mediated posttranscriptional regulation of p21 is involved in the effect of Glycyrrhiza uralensis licorice aqueous extract on polyamine-depleted intestinal crypt cells proliferation.

Glycyrrhiza uralensis licorice has long been used worldwide as a food additive and herbal medicine. It possesses a remarkable healing action on gastrointestinal ulcers. The present study was carried out to assess the effect of licorice on intestinal crypt cell proliferation and to investigate the corresponding molecular mechanism. Considering the role of crypt stem cells in intestinal mucosa repair, a well-established cytostatic cellular model, polyamine-depleted IEC-6 cells, was utilized to evaluate the effect of aqueous licorice on the proliferation of intestinal crypt cells. The growth inhibition of IEC-6 cells caused by alpha-difluoromethylornithine could be significantly reversed by concomitant treatment with 40 µg/ml and 80 µg/ml licorice aqueous extract. In particular, the restoration of cell cycle progression was accompanied by a decrease in p21 mRNA level and cytoplasmic accumulation of the RNA-binding protein HuR, which was shown to be involved in the destabilization of p21 mRNA. Using a biotin pull-down assay and a luciferase assay, it was found that licorice-modulated p21 mRNA expression was achieved by HuR-targeted AU-rich and U-rich elements that resided in the 3' untranslated region of p21 mRNA. These results demonstrate that licorice can exert its action on stimulating the growth of intestinal crypt cells by regulating p21 mRNA level at the posttranscriptional level by HuR.

Polyamines mediate glutamine-dependent induction of the intestinal epithelial heat shock response.

Heat shock proteins (Hsps) are highly conserved proteins that play a role in cytoprotection and maintaining intestinal homeostasis. Glutamine is essential for the optimal induction of intestinal epithelial Hsp expression, but its mechanisms of action are incompletely understood. Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. Thus we investigated whether polyamines (putrescine, spermidine, or spermine) and their precursor ornithine mediate the induction of Hsp expression in IEC-18 rat intestinal epithelial cells. As previously observed, glutamine was required for heat stress induction of Hsp70 and Hsp25, although it had little effect under basal conditions. Under conditions of glutamine depletion, supplementation of ornithine or polyamines restored the heat-induced expression of Hsp70 and Hsp25. When ODC was inhibited by α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress induction of Hsp70 and Hsp25 was decreased significantly, even in the presence of glutamine. Ornithine, polyamines, and DFMO did not modify the nuclear localization of heat shock transcription factor 1 (HSF-1). However, DFMO dramatically reduced glutamine-dependent HSF-1 binding to an oligonucleotide with heat shock elements (HSE), which was increased by glutamine. In addition, exogenous polyamines recovered the DNA-binding activity. These results indicate that polyamines play a critical role in the glutamine-dependent induction of the intestinal epithelial heat shock response through facilitation of HSF-1 binding to HSE.

Effect of dietary green tea extract and aerosolized difluoromethylornithine during lung tumor progression in A/J strain mice.

Chemoprevention strategies to prevent the development of lung cancer in at-risk individuals are a key component in disease management. In addition to being highly effective, an ideal chemopreventive agent will require low toxicity as patients are likely to require treatment for several years before their risk of cancer is lowered to background levels. In principle, a combination of safe agents that work through distinct mechanisms will improve efficacy while simultaneously maintaining a favorable safety profile. Here, we describe the use of the decaffeinated green tea extract Polyphenon E (Poly E) (1% in diet) and aerosolized difluoromethylornithine (DFMO) (20 mg/kg/day, 5 days/week) in a mouse lung cancer chemoprevention study using a progression protocol. Female A/J mice were injected with benzo[a]pyrene (B[a]P) at 8 weeks of age and precancerous lesions allowed to form over a period of 21 weeks before chemoprevention treatment for an additional 25 weeks. Poly E treatment did not significantly inhibit average tumor multiplicity but reduced per animal tumor load. Analysis of tumor pathology revealed a specific inhibition of carcinomas, with the largest carcinomas significantly decreased in Poly E-treated animals. Aerosolized DFMO did not have a significant effect on lung tumor progression. Magnetic resonance imaging of B[a]P-induced lung tumors confirmed the presence of a subset of large, rapidly growing tumors in untreated mice. Our results suggest a potential role for green tea extracts in preventing the progression of large, aggressive lung adenocarcinomas.

Spermine-induced morphogenesis and effect of partial immersion system on the shoot cultures of banana.

Contribution of exogenous polyamines (PAs) and polyamine-inhibitors on plantlet regeneration patterns of banana (cv. Nanjanagudu Rasabale-AAB) was studied and the performance of regenerated shoots in temporary immersion system was evaluated. The rhizome explants (without shoot bud) of in vitro shoots produced a mixture of embryogenic and nonembryogenic calli on modified MS medium. The analyses of endogenous pools of polyamines showed higher levels of PAs in embryogenic than in nonembryogenic calli. Supplementation of various levels of (10-50 microM) spermine (Spm), spermidine (Spd), and putrescine (Put) to cultures with secondary embryogenesis showed that about 50% of embryogenic calli rapidly produced secondary embryos only in the presence 40 microM Spm but not in other treatments. The crucial role of Spm was further confirmed by the use of 0.1 mM each of alpha-DL-Difluromethylornithine and alpha-DL-Difluromethylarginine along with Spm where the presence of inhibitors concomitantly inhibited the secondary embryogenesis. The shoots obtained from the embryogenic cultures were checked for their performance on solid medium (SM) and partial immersion system (PIS). The rate of shoot multiplication was higher in PIS than in SM throughout 6 weeks culture period. Uniformity in elongation of all the shoot buds was observed in PIS but not in SM. Evaluation for the acclimatization, survival under greenhouse conditions revealed the better performance of PIS-derived plants than those from SM.

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Functional consequences of polyamine synthesis inhibition by L-alpha-difluoromethylornithine (DFMO): cellular mechanisms for DFMO-mediated ototoxicity.

L-Alpha-difluoromethylornithine (DFMO) is a chemopreventive agent for colon cancer in clinical trials. Yet, the drug produces an across-frequency elevation of the hearing threshold, suggesting that DFMO may affect a common trait along the cochlear spiral. The mechanism for the ototoxic effects of DFMO remains uncertain. The cochlear duct is exclusively endowed with endocochlear potential (EP). EP is a requisite for normal sound transduction, as it provides the electromotive force that determines the magnitude of the receptor potential of hair cells. EP is generated by the high throughput of K(+) across cells of the stria vascularis, conferred partly by the activity of Kir4.1 channels. Here, we show that the ototoxicity of DFMO may be mediated by alteration of the inward rectification of Kir4.1 channels, resulting in a marked reduction in EP. These findings are surprising given that the present model for EP generation asserts that Kir4.1 confers the outflow of K(+) in the stria vascularis. We have proposed an alternative model. These findings should also enable the rational design of new pharmaceuticals devoid of the untoward effect of DFMO.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Ischemia-reperfusion selectively impairs nitric oxide-mediated dilation in coronary arterioles: counteracting role of arginase.

A reduction in L-arginine availability has been implicated in the impairment of endothelium-dependent nitric oxide (NO)-mediated vasodilation by ischemia-reperfusion (I/R). However, the mechanisms contributing to dysregulation of the L-arginine pool remain unknown. Because endothelial cells can metabolize L-arginine via two major enzymes, that is, NO synthase (NOS) and arginase, we hypothesized that up-regulation of arginase during I/R reduces L-arginine availability to NOS and thus impairs NO-mediated vasodilation. To test this hypothesis, a local I/R was produced in the porcine heart by occlusion of a small branch of left anterior descending artery for 30 min, followed by reperfusion for 90 min. Arterioles (60-110 microm) isolated from non-ischemic and ischemic regions of subepicardium were cannulated and pressurized without flow for in vitro study. Vessels from both regions developed similar levels of basal tone. Although the dilation of I/R vessels to endothelium-independent agonist sodium nitroprusside was not altered, the endothelium-dependent NO-mediated dilations to adenosine and serotonin were attenuated. I/R not only inhibited arteriolar production of NO but also increased arteriolar arginase activity. Arginase inhibitor alpha-difluoromethylornithine enhanced NO production/dilation in normal vessels and also restored the NO-mediated function in I/R vessels. Treating I/R vessels with L-arginine also restored vasodilations. Immunohistochemical data revealed that I/R up-regulated arginase but down-regulated NOS expression in the arteriolar endothelium. Pretreating the animals with protein synthesis inhibitor cycloheximide prevented I/R-induced arginase up-regulation and also preserved NO-mediated vascular function. These results suggest that one mechanism by which I/R inhibits NO-mediated arteriolar dilation is through increased arginase activity, which limits the availability of L-arginine to NOS for NO production. In addition, the inability of arginase blockade or L-arginine supplementation to completely restore vasodilatory function may be attributable to the down-regulation of endothelial NOS expression.

Alpha-difluoromethylornithine, ornithine decarboxylase inhibitor, antagonizes H2O2-induced cytotoxicity in HL-60 leukemia cells: regulation of iron-dependent lysosomal damage.

Recent studies indicate that reactive oxygen species, such as H2O2, can be generated by anti-cancer drugs, can damage cells, and then induce apoptotic cell death. In this study, we reported whether polyamines were capable of affecting apoptotic cell death triggered by H2O2 in leukemia cells or not. Alpha-difluoromethylornithine treatment (DFMO, 3 mmol/L, 48 h), which depletes intracellular putrescine by inhibiting ornithine decarboxylase, reduced H2O2-induced cell death in the HL-60 leukemia cells. Cytotoxicity caused by H2O2 in putrescine-depleted cells was 50% lower than that in the control cells, as determined by propidium iodide, the annexin V and DNA fragmentation assays. Following putrescine (1 mmol/L) supplement, cell death induction caused by H2O2 was restored to a similar level as the DFMO-untreated control cells. It seems that this partly resulted from the intralysosomal iron-dependent oxidation of the cells because DFMO did not significantly affect the increment of enzymes related to oxidative-stress resistance. Putrescine depletion by DFMO treatment reduced the cellular iron uptake of the cells by about 70%. In parallel to the reduction of iron uptake, lysosomal damage (assayed by acridine orange relocalization or uptake test) in the DFMO-treated cells was far less than that in the control cells. Moreover, putrescine supplement also restored the iron uptake to the control cell levels. Pre-incubation with desferrioxamine (DFO), which chelates iron and forms a non-reactive Fe-DFO complex that is localized in the lysosomal compartment, inhibited H2O2-induced cell death. This work suggests that polyamines may play a critical role in apoptotic cell death triggered by H2O2 via the regulation of the iron-dependent instability of the lysosome.